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The Egyptian Journal of Hospital Medicine (Sep. 2010) Vol., 40 : 314 - 334
-314-
Influence of Ionizing Radiation on Echis pyramidium Snake Venom:
Biochemical and Immunological Aspects
*Adel G. El-Missiry, ***Esmat A. Shaban, **Mohamed R. Mohamed, *Ahmed A.
Ahmed, **Nadia M. Abdallah and *Manal I. Moustafa
*Medical Research Center – faculty of medicine Ain Shams University.
** Department Of biochemistry – Faculty of Science Ain Shams University
***Department of Drug Radiation Research – National Center for Radiation Research
and Technology – Atomic Energy Authority
Abstract
The effect of a single LD50 dose of native Echis pyramidum venom (27.69µg/mouse) on the activities of certain serum enzymes levels: aspartate aminotransferase (AST), alanine
aminotransferase (ALT), alkaline phosphatase (ALP), urea, creatinine, lactate dehydrogenase
(LDH), creatine phosphokinase (CPK), creatine kinase isoenzyme (CK-MB) were studied.
Samples from the serum were collected 4hr following LD50 venom dose intraperitonealy injected in male Swiss albino mice. The activities of these enzymes showed significant elevation
compared to the non-envenomated group. In contrast, an equivalent dose of 1.5 kGy γ irradiated
Echis pyramidum venom (27.69g/mouse) did not cause any significant increase compared to
non-envenomated group.
The effect of a dose that is equivalent to ½ LD50 (13.8 µg/50 µl) of native Echis pyramidum
venom on plasma creatine phosphokinase (CPK) induced a significant increase of creatine phosphokinase (CPK) level compared to normal control (P<0.01). In contrast, an equivalent
dose of 1.5 kGy γ irradiated Echis pyramidum venom showed non significant difference in
creatine phosphokinase activity when compared to the normal control. Light microscopic examinations of gastrocenemius muscles of mice injected with native Echis pyramidum venom
(½ LD50; 13.8µg/50µl) showed fragmentation, disorganization, loss of myofibrils in some of the
muscle fibers, hemorrhage in-between the muscle fibers and mononuclear cellular infiltration. While light microscopic examinations of gastrocenemius muscles of mice injected with 1.5 kGy
γ irradiated Echis pyramidum venom (13.8µg/50µl; a dose identical to that used from native
venom) showed that most muscle fibers were of normal appearance except for small area of
fragmentation and disorganized myofibrils and oedema of the intercellular connective tissue. Double immunodiffusion test revealed a similar reactivity for native, 1 kGy, 1.5 kGy and 3 kGy
γ irradiated Echis pyramidum venoms against a commercial polyvalent Egyptian antivenin. The
visible lines obtained in the immunodiffusion reactions were identical and joined smoothly at the corners, indicating that there was no change in their antigenic reactivity. These results
demonstrate that the ability of the venom antigens to react with its corresponding antibodies was
maintained in spite of being exposed to radiation doses of 1 kGy, 1.5 kGy and 3 kGy. Both antivenins raised against native or 1.5 kGy γ irradiated venoms recognized Echis
pyramidum venom when submitted to protein blotting, but the anti 1.5 kGy γ irradiated venom
show a higher intensity bands than the antivenin raised against native Echis pyramidum venom,
in spite of having less neutralizing activity (native neutralize 50 LD50, 1.5 kGy γ irradiated neutralize 40 LD50), this indicates that antibodies were formed not only for toxic fraction but
also for non toxic fraction.
Irradiation of the whole Echis Pyramidium Venom with 1.5KGy reduced its lethality 12.5 times though keeping its immunogenicity. The 1.5KGy dose was shown to be the best radiation dose
to promote detoxification without significantly affecting its immunogenicity. Thus results of this
study confirm the conclusion that γ radiation is a suitable way to detoxify Echis Pyramidium
Venom without affecting its immunogenicity provided that proper dose is used.
Influence of ….
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Keywords: Echis pyramidum, γ irradiated venom, aspartate aminotransferase, alanine
aminotransferase, alkaline phosphatase, urea, creatinine, lactate dehydrogenase, creatine
phosphokinase, creatine kinase isoenzyme, Double immunodiffusion, protein blotting.
Introduction There are variations in the
pharmacological, antigenic and biochemical
properties of the different snake venoms,
both between species and within a single species due to geographic and ontogenetic
variables (S′anchez et al., 1992).
According to Aguiyi et al. (2001), the effect of lethal Echis carinatus venom on
serum enzyme levels and blood plasma
coagulation parameters in rats subjected to intraperitoneal (i.p) venom injection,
measurements of the enzyme and
coagulation parameter levels 4h after
venom administration showed an increase in the level of enzymes; lactate
dehydrogenase (LDH), ALT and creatinine
kinase (CK) as well as a change in the level of coagulation parameters D-Dimer
and Quick due to envenomation.
Also, it has been reported that treatment of
Swiss albino rats with a sublethal dose of Cearates cearastes venom (0.75 μg/g body
weight) increased significantly the activities
of lactate dehydrogenase, isocitrate dehydrogenase and aldolase in the serum,
liver and skeletal muscle 0.5–6 h
postenvenomation (Abu-Sinna et al., 1992). Moreover, this treatment induced
hypoglycemia after 15 min which persisted
for 24 h (Abu-Sinna et al., 1993) and
increased the plasma total protein level significantly, while it decreased the liver
and skeletal muscle protein contents 15–24
h after envenomation (Abdel-Aal et al., 1992).
Artashes and Silva (2006) reported that
venom of the Armenian adder (Vipera
raddei Boettger, 1890) was tested for its
ability to induce histopathological changes
in rabbits after long-term (once every 6
days for 30 days) intramuscular injection of the venom (0.35 mg/kg b.w.; approx. 0.5
LD50), induced moderate histopathological
changes in vital organs (liver, heart, kidney, adrenal, lung and spleen).
In Brazil, gamma rays have been employed
to detoxify Brazilian snake venoms in order
to improve antivenin production. Murata et al. (1990) irradiated Crotalus durissus
terrificus venom with gamma rays using
different doses and found that 2,000 Gy
was a good compromise in irradiation dosage for venom solutions, which
promoted significant venom detoxification,
yet maintained many of the venom original immunological properties. In 2000,
Gallacci et al. studied other snake venoms
such as Bothrops jararacussu and Lachesis
muta venoms and carried out some
experiments using bee venom with good
results of detoxification when subjected to
the effects of gamma rays. In 2003, Bennacef-Heffar and Laraba-
Djebari studied the effect of gamma
irradiation on the venom of Vipera lebetina (one of the two widespread snakes in
Algeria). Vipera lebetina venom was
irradiated with two doses of gamma rays (1
and 2 kGy) from a 60
Co source, and the venom's toxic, enzymatic, and structural
properties were analyzed. The
immunogenic properties of the irradiated venom were preserved and the antisera
obtained were able to neutralize the toxic
effect of Vipera lebetina native venom. These results indicate that irradiation of
Vipera lebetina venom with a dose of 2
kGy can promote a significant
detoxification while keeping the immunological properties intact.
In 2002, Souza et al., investigated the
ability of gamma radiation from 60
Co (2000 Gy) to attenuate the toxic effects of
Bothrops jararacussu venom on mouse
neuromuscular preparations in vitro. These findings support the hypothesis that gamma
irradiation could be an important tool to
improve antisera production by reducing
toxicity while preserving immunogenicity. It was reported recently that intravenous
administration of antivenin, prepared from
IgG of venom immunized horses or sheep, is the only effective treatment of systemic
envenoming (Harrison et al., 2006).
Adel El-Missiry ….et al
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It has been suggested in the past that
detoxified venoms can be used to produce antiserum as an effort to protect the animals
from the venom toxicity. It is therefore
important to be sure that the detoxified venoms do not lose their immunogenicity.
Several techniques have been used to
detoxify venoms, such as mixing the venom
with carboxymethylcellulose (Moroz et al., 1963), irradiation by gamma rays (Baride et
al., 1980), adding formaldehyde (Costa et
al., 1985), controlled iodination (Daniel et al., 1987) and encapsulation of purified
toxins in liposomes (Freitas and Fr ezard,
1997). One method that has been shown to
be effective for attenuating venom toxicity and maintaining immunogenicity is γ
irradiation (Nascimento et al., 1996).
Proteins irradiated, either in dry state or in solution, undergo chemical and
physicochemical changes that can alter their
primary, secondary and tertiary structures, while keeping many of their native
immunological properties intact ( Skalka
and Antoni, 1970).
In this study the effects of gamma
irradiation on the venom of Echis
pyramidum was determined. Venom was irradiated with 1.5KGy and 3KGy from 60
Co source and the venom's toxic,
enzymatic and immunological properties
were analyzed.
Material and methods
- Venom Venom used in this study was that of the
Egyptian Viper Echis pyramidum and was
obtained from Laboratory Animal Unit of Medical Research Center, Faculty of
medicine, Ain Shams University.
- Antivenin
Egyptian polyvalent antivenin obtained from the Egyptian Organization of
Biological products and Vaccines, Agouza,
Cairo, Egypt, was used. The lyophilized polyvalent antivenin produced in horses
was kept at 4ºC and reconstituted with
saline (20mg / ml) before use.
- Irradiation facilities The Echis pyramidum venom was irradiated with 1 kGy, 1.5 kGy and 3 kGy γ
rays in the National Center for Radiation
Research andTechnology, Cairo, Egypt, using cobalt-60 gamma cell 220,
manufactured by the atomic energy of
Canada (AECL). The radiation dose rate
was 1.4 Rad per second. Throughout this study 45 males Swiss
albino mice weighing 20-25 gm were
allocated to the following groups: 1- Control (non envenomated) group
(n=15). Received 0.5 ml intraperitoneal
injection of saline.
2- Native Echis pyramidum venom envenomated group (n=15). Received a
dose of 27.7 µg (in 0.5 ml saline) per
mouse. 3- 1.5 kGy γ irradiated Echis pyramidum
venom envenomated group (n=15).
Received dose of 27.7 µg (in 0.5 ml saline) per mouse.
Native and 1.5 kGy γ irradiated Echis
pyramidum venom (27.7g) dissolved in
0.5ml of saline, was injected (i.p.) into two
groups of 15 mice each. Control group
received 0.5ml i.p. injection of saline. After 4h, blood samples were collected and
centrifuged to obtain serum.
- Lethality
The LD50 of native and irradiated venoms were determined according to the method
of Reed and Muench (1938), using male
albino Swiss mice 20-25 gm. Ascending concentrations of 5 dose levels of the
freshly prepared venom solutions were
arranged in a geometric progression by using increasing factor equal 1.2 starting by
a dose which kills approximately 0-10 % of
the animals and ending by a dose which
kills approximately 90-100 % of the injected animals. Each dose level was tested
in 5 mice, and all injections were given
intraperitoneally, and deaths or survivals of envenomated animals were recorded after
24 hs from the time of the injection.
- Preparation of antivenin in rabbits
Adult male rabbits weighing 3-3.5 Kg were maintained under standard
conditions of boarding and given standard
food were divided into 2 groups:
Influence of ….
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1- Rabbits received native intradermal
multisits injection of Echis Pyramidum venom (n=3)
2- Rabbits received intradermal multisits
injection of 1.5 kGy γ irradiated Echis
Pyramidum venom (n=3).
Immunization was done in the presence or absence of complete (CFA) or incomplete
(IFA) Freund’s adjuvant according to table
1.
Table (1): Immunization schedule for rabbits using either native or 1.5 kGy γ irradiated Echis
pyramidum venom.
Week Native
venom(µg)
1.5 kGy γ irradiated
venom
(µg)
Freund’s adjuvant
0 500 1000 CFA
2 500 1000 CFA
4 1000 2000 IFA
6 1200 2400 -
8 1200 2400 -
10 1500 3000 - By the end of this schedule serum was obtained from the two rabbit groups and tested for
Protein blotting .
Measured parameters
Determination of serum ALT and AST
activities were carried out by (Reitman and Frankel, 1957). Serum alkaline phosphatase
activity (ALP) was determined in serum.
The ALP was performed kinetically using a test reagent kit according to the method of
Rec. GSCC,(1972). Determination of serum
urea was carried out by (Patton and Crouch,
1977), serum creatinine activity was carried out by using the method of (Henry,
1974), serum LDH was carried out by using
the method of (IFCC, 1980), serum (CPK-MB) was carried out according to the
methos of Rec. GSCC. (1978), Serum
creatine kinase isoenzyme (CK-MB) was carried out by an immunoinhibition method
(Szasz and Busch, 1979)
- Myotoxic activity.
Myotoxic activity of the native and 1.5 kGy γ irradiated Echis pyramidum venom was
determined in mice according to Yongming
Bao et al., (2005). Throughout this study 12 male albino Swiss
mice weighing 20-25 gm were allocated in
the following groups:
1- Control group: Untreated non
envenomated mice only received 0.5ml intraperitoneal injection of saline.
2- Native Echis pyramidum venom
envenomated group.
3- 1.5 kGy γ irradiated Echis pyramidum
venom envenomated group. Native and 1.5 kGy γ irradiated Echis
pyramidum venom ½ LD50 (13.8µg), was
dissolved in 50µl of saline and injected into
two groups of four mice each in their left gastrocenemius muscle. A control group of
4 mice received 50µl of saline. After 3h,
blood samples were collected and creatine phosphokinase (CPK) levels in plasma were
determined.
- Histological changes
In addition to CPK activity measurements, formalin fixed muscle tissue samples were
obtained after 3h, and processed for
histological evaluation of muscle damage. Sections of the muscles were Dehydrated
and cleared in ascending grades of ethyl
alcohol and xylene respectively. The specimens are then embedded in paraffin
wax. Sections of 5 microns thickness were
cut and stained with haematoxyline and
eosin and examined under light microscope
Adel El-Missiry ….et al
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according to the method of Drury and
Wallington (1976). - Double immunodiffusion
Immunodiffusion experiments were carried
out as originally described by Ouchterlony (1945).
- Protein blotting
Western Blot method described by Towbin
et al. (1979) which results in efficient and reproducible electrophoretic protein transfer
from polyacrylamide gels onto
polyvinylidene difluoride (PVDF) membrane, was applied.
Statistical Analysis of Data:
The statistical analysis was performed using student t – test by Prism Dimo- program
and origin 6.1. the method used for the
analysis of the results is that given by Millon et al., (1986)
Results
1- Effect of native and 1.5kGy γ
irradiated Echis pyramidum venoms on
liver tests:
(tab.2, fig. 1) show the differences in the
mean values for ALT, AST and ALP among the different groups. These results
demonstrated that i.p injection of an LD50
dose (27.69 g/mouse) of native Echis
pyramidum venom induced a highly
significant increase in ALT, AST and ALP
compared to non-envenomated control (P<0.001).
In contrast, an equivalent dose of 1.5 kGy γ
irradiated Echis pyramidum venom (27.69
g/mouse) did not cause any significant
change in ALT, AST or ALP compared to
the normal non-envenomated control. Moreover, the increase in the level of ALT,
AST and ALP in case of intraperitoneal
injection of native Echis pyramidum venom was highly significant compared to
intraperitoneal injection of 1.5 kGy γ
irradiated Echis pyramidum venom (P<0.001).
2- Effect of native and 1.5 kGy γ
irradiated Echis pyramidum venoms on
kidney tests: (Tab. 3, fig. 2) show the differences in the
mean values for urea and creatinine among
the different groups. These results
demonstrated that i.p injection of an LD50 dose (27.69µg/mouse) of native Echis
pyramidum venom induced a highly
significant increase in urea and creatinine compared to non-envenomated control
(P<0.001).
In contrast, an equivalent dose of 1.5 kGy γ
irradiated Echis pyramidum venom (27.69
g/mouse) did not cause any significant
change in urea or creatinine compared to non-envenomated control. Moreover, the
increase in the level of urea and creatinine
in case of i.p injection of native Echis
pyramidum venom was highly significant compared to that in case of i.p injection of
1.5 kGy γ irradiated Echis pyramidum
3- Effect of native and 1.5 kGy γ
irradiated Echis pyramidum venoms on
heart tests: (tab. 4, fig. 3) show the differences in the
mean values for LDH, CPK and CK-MB
among the different groups. These results
demonstrated that i.p injection of an LD50
dose (27.69 g/mouse) of native Echis
pyramidum venom induced a highly significant increase in LDH, CPK and CK-
MB compared to non-envenomated control
(P<0.001).
In contrast, an equivalent dose of 1.5 kGy γ irradiated Echis pyramidum venom (27.69
g/mouse) did not cause any significant
increase in LDH, CPK or CK-MB
compared to non-envenomated control.
Moreover, the increase in the level of LDH,
CPK and CK-MB in case of i.p injection of native Echis pyramidum venom was highly
significant compared to that in case of i.p
injection of 1.5 kGy γ irradiated Echis
pyramidum venom (P<0.001).
4- Myotoxic activity:
(Tab. 5 , fig 4) show the effect of a dose
that is equivalent to ½ LD50 (13.8 µg/50 µl) of native Echis pyramidum venom on
plasma creatine phosphokinase (CPK)
levels. Native Echis pyramidum venom induced a significant increase of creatine
phosphokinase (CPK) of the
gastrocenemius muscles compared to normal control (P<0.01). In contrast, an
equivalent dose of 1.5 kGy γ irradiated
Influence of ….
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Echis pyramidum venom did not show any
significant increase of creatine phosphokinase when compared to control.
Moreover, the observed increase in CPK
following injection of native Echis
pyramidum venom was significantly higher
than that observed following injection of
the 1.5 kGy γ irradiated venom (P<0.01).
5- Histological changes Fig. (5) shows a light microscopic
examination of gastrocenemius muscles of
mice injected with saline (normal control). It shows bundles of skeletal muscle fibers
with evident cross striations and
peripherally situated multiple nuclei.
Fig. 6 (A) and (B) show light microscopic examinations of gastrocenemius muscles of
mice injected with native Echis pyramidum
venom (½ LD50; 13.8µg/50µl). They show fragmentation, disorganization, loss of
myofibrils in some of the muscle fibers,
hemorrhage in-between the muscle fibers and mononuclear cellular infiltration.
Fig. 7 (A) and (B) show light microscopic
examinations of gastrocenemius muscles of
mice injected with 1.5 kGy γ irradiated
Echis pyramidum venom (13.8µg/50µl; a
dose identical to that used from native venom). They show most of the muscle
fibers were of normal appearance except for
small area of fragmentation and disorganized myofibrils and oedema of the
intercellular connective tissue.
6- Double immunodiffusion
The immunogenic reactivity of the native, 1, 1.5 and 3 kGy γ irradiated Echis
pyramidum venoms to commercial horse
polyvalent Egyptian antivenin was tested using the double immunodiffusion method
fig. (8). All tested venoms showed similar
reactivity. The visible lines obtained in the
double immunodiffusion reactions were identical and joined smoothly at the
corners, indicating that there was no change
in the antigenic determinants.
7- Western blotting analysis
By analyzing the Western blotting profiles
Fig. (9), it was observed that the antivenin raised against the native and the 1.5 kGy γ
irradiated Echis pyramidum venom
recognized equally all the bands present on
the native Echis pyramidum venom.
Table (2): The mean values of serum levels of alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) for normal control, native and 1.5 kGy γ
irradiated Echis pyramidum venom (27.69µg/mouse) envenomated groups (4hr after i.p
injection of mice), (n = 15).
Groups
Parameters
Control
Native venom
1.5 kGy
ALT (U/L)
Mean ± S.E.
% change
P Value
P1 Value
59.1 ± 3
161.7 ± 4.9
+ 173.6
P < 0.001**
67.4 ± 2.7
+ 14
N.S.
P < 0.001**
AST (U/L)
Mean ± S.E.
% change
P Value
P1 Value
228.3 ± 4.7
377.8 ± 9
+ 65.4
P < 0.001**
238.1 ± 4
+ 4.2
N.S.
P < 0.001**
ALP (U/L) Mean ± S.E.
% change
P ValueP1 Value
119.1 ± 5.1
204 ± 7.9
+ 71.3
P < 0.001**
129.8 ± 4.9
+ 8.9
N.S.
P < 0.001**
P: Statistical difference from normal control. P1: Statistical difference from native venom.
P <0.001: highly significant. N.S.: Non significant.
Adel El-Missiry ….et al
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Table (3): The mean values of serum levels of urea and creatinine for normal control, native
and 1.5 kGy γ irradiated Echis pyramidum venom (27.69µg/mouse) envenomated groups (4hr after i.p injection of mice), (n = 15).
Groups
Parameters
Control Native venom 1.5 kGy
Urea (mg/dl)
Mean ± S.E.
% change
P Value
P1 Value
32.7 ± 1.4
78.5 ± 4
+ 140
P < 0.001**
35.8 ± 1.5
+ 9.4
N.S.
P < 0.001**
Creatinine(mg/dl)
Mean ± S.E.
% change
P Value
P1 Value
0.76 ± 0.02
1.5 ± 0.03
+ 97.3
P < 0.001**
0.83 ± 0.03
+ 9.2
N.S.
P < 0.001**
P: Statistical difference from normal control. P1: Statistical difference from native venom.
P <0.001: highly significant. N.S.: Non significant.
Table (4): The mean values of serum levels of lactate dehydrogenase (LDH), creatine kinase
(CK) and creatine kinase isoenzyme (CK-MB) for normal control, native and 1.5 kGy γ
irradiated Echis pyramidum venom envenomated groups (4hr after i.p injection of mice), (n = 15).
Groups
Parameters
Control
Native venom
1.5 kGy
LDH (U/L)
Mean ± S.E.
% change
P Value
P1 Value
1672.5 ± 28.4
2680.8 ± 40.4
+ 60.3
P < 0.001**
1736.4 ± 31.3
+ 3.8
N.S.
P < 0.001**
CK (U/L)
Mean ± S.E.
% change
P Value
P1 Value
670 ± 15.4
1135.6 ± 19
+ 69.5
P < 0.001**
719.5 ± 12.7
+ 7.3
N.S.
P < 0.001**
CK-MB (U/L)
Mean ± S.E.
% change
P Value
P1 Value
161.2 ± 3.3
282.3 ± 6.7
+ 75.1
P < 0.001**
170.9 ± 2.4
+ 6
N.S.
P < 0.001**
P: Statistical difference from normal control. P1: Statistical difference from native venom.
P <0.001: highly significant. N.S.: Non significant.
Influence of ….
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Fig. (1): The mean values of serum levels of alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) for normal control and native and 1.5 kGy
γ irradiated Echis pyramidum venom envenomated groups (4h after i.p injection of mice), (n =
15). Each column represents the mean value of fifteen mice. **: Highly significant difference from normal control.
**: Highly significant increase from normal control.
Fig. (2): The mean values of serum levels of urea and creatinine for normal control, native and
1.5 kGy γ irradiated Echis pyramidum venom envenomated groups (4hr after i.p injection of mice). Each column represents the mean value of fifteen mice.
**: Highly significant difference from normal control.
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**
**
**
0
500
1000
1500
2000
2500
3000
Mea
n (U
/L)
1 2 3
LDH CK CK-MB
Control Native venom 1.5 kGy
Fig. (3): The mean values of serum levels of lactate dehydrogenase (LDH), creatine kinase
(CK) and creatine kinase isoenzyme (CK-MB) for normal control, native and 1.5 kGy γ irradiated Echis pyramidum venom envenomated groups (4hr after i.p injection of mice). Each
column represents the mean value of fifteen mice.
**: Highly significant increase from normal control.
Table (5): Myotoxic activities of native and 1.5 kGy γ irradiated Echis pyramidum venoms.
Plasma creatine kinase (CK) activity was determined 3hr after the intramuscular injection of the
native or 1.5 kGy γ irradiated venom (13.8µg/50µl) in the gastrocenemius muscle of mice (n = 4). n: Number of mice in each group.
Groups
Plasma
(CPK U/L)
Control
Native venom
1.5 kGy
Mean ± SE 1039 ± 162.4 4035.2 ± 411.8 1885 ± 217.9
P Value / control P <0.01* N.S.
P <0.01: significant. N.S.: Non significant.
Fig. (4): Myotoxic activities of native and 1.5 kGy γ irradiated Echis pyramidum venoms.
Plasma creatine kinase (CK) activity was determined 3hr after the intramuscular injection of the
native or 1.5 kGy γ irradiated venom (13.8µg/50µl) in the gastrocenemius muscle of mice (n = 4).
*: Significant increase from normal control.
Influence of ….
- 323 -
Fig. (5): A photomicrograph of a section of the gastrocenemius muscle of control mice showing
bundles of skeletal muscle fibers with evident cross striations (↑). Notice the peripherally situated multiplenuclei (↑↑), X 640.
(A) (B)
Fig. (6): A photomicrograph of a section of the gastrocenemius muscle of mice treated with native Echis pyramidum venom (13.8µg/50µl).
(A) showing fragmentation of muscle fibers (↑). Notice hemorrhage in-between the muscle
fibers (↑↑) and mononuclear cellular infiltration (∆), X 250.
(B) showing disorganization and loss of myofibrils in some of the muscle fibers (↑). Notice
hemorrhage in-between the muscle fibers (↑↑) and mononuclear cellular infiltration (∆), X 640.
Adel El-Missiry ….et al
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(A) (B)
Fig. (7): A photomicrograph of a section of the gastrocenemius muscle of mice treated with 1.5 kGy γ irradiated Echis pyramidum venom (13.8µg/50µl).
(A) showing most of the muscle fibers were of normal appearance except for small area of fragmentation (↑). Notice the oedema of the intercellular connective tissue (↑↑), X 250.
(B) showing most of the muscle fibers were of normal appearance with evident cross striations (↑). Notice small area of disorganized myofibrils (↑↑) with intercellular connective tissue
oedema (∆), X 640.
Fig. (8): The immunogenic reactivity of horse polyvalent antivenin (in the central well) against native, 1, 1.5 and 3 kGy γ irradiated Echis pyramidum venoms (20 mg/ml).
Influence of ….
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1 2 3
Fig. (9): Western blotting of antivenin raised against native and 1.5 kGy γ irradiated Echis
pyramidum venom: samples of 20µg of native Echis pyramidum venom (column 1) were
submitted to Western blotting and revealed by antivenin raised against native (column2) and
antivenin raised against 1.5 kGy γ irradiated Echis pyramidum venom (column 3).
Discussion: The performed experiment assessed the
impact of γ irradiation of Echis pyramidum
venom on the effects induced by the venom on some biochemical markers. The present
work showed the effects of LD50
(27.69µg/mouse) of native Echis
pyramidum venom on some biochemical parameters.
The activities of Aspartate aminotransferase
(AST), Alanine aminotransferase (ALT) and Alkaline phosphatase (ALP) underwent
a highly significant increase following
envenomation with native Echis
pyramidum venom compared to the normal
control. The results are in agreement with
prior reports. Sant et al. (1974) and Tembe
et al. (1975) by using Echis Carinatus snake venom found that there were
elevations in the serum concentration of
AST, ALT and ALP in animals four hours
post envenomation in comparison with the
control group. Also, it was reported by
Abdel - Nabi and Rahmy (1992), using Echis carinatus venom, that injection of
sublethal dose caused a significant rise in
serum AST, ALT and ALP in rats accompanied with disturbances in the
hepatic and renal functions of the
envenomated animals through severe hepatocellular injuries, necrosis of
hepatocytes and kidney tubules as well as
nephrotoxic action. Moreover, Shaban and
Hafez (2003) reported that intraperitoneal injection of a sublethal dose of Naja haje
venom (0.2mg/kg) in rats induced a
significant elevation in the activities of AST, ALT and ALP as compared to normal
control. Furthermore, Abdel-Nabi (1993)
reported that the sublethal dose of both crude venom and its B fraction injection
caused a significant elevation in AST, ALT
and Alkaline phosphatase (ALP) activities.
Adel El-Missiry ….et al
- 326 -
This elevation in the ALP could be
attributed either to the destruction of the liver, kidney and/or heart tissue. Also
elevation in AST and ALT, gives evidence
about the destruction of the hepatocytes as a result of venom injection. Also Mohamed et
al, (1981) reported that Bitis arietans
venom significantly increased AST and
ALT levels in serum while decreased them in liver, heart and kidney. Similarly,
alkaline phosphatase levels increased in
serum and decreased in heart and liver. As for the effect on the kidney and renal
functions, the LD50 (27.69µg/mouse) of
native Echis pyramidum venom induced a
highly significant increase in urea and creatinine concentrations compared to the
normal control.
The recorded results are in accordance with those found by Abdel Nabi 1993 who has
reported that a sublethal dose of both crude
Cerastes cerastes venom and its B fraction showed a significant rise in blood urea
nitrogen and this was concomitant to a
significant increase in serum creatinine
levels as well. The increase in these values is used as an indicator of renal failure.
In other studies on the effect of the viper
Cerastes cerastes cerastes venom on the kidney, Abdel - Aal ( 1998 ) reported that
viper venom increased serum creatinine and
urea significantly, 30 min. following injection, and that this effect persisted for
up to 7 days, indicating renal failure. Also,
Tu (1991) and Merchant et al. (1989),
reported that renal diseases caused by various snake venoms were characterized
by raised urea , creatinine and potassium in
oliguric patients. Moreover, Shaban and Hafez (2003)ا reported that intraperitoneal
injection of a sublethal dose of Naja haje
venom (0.2mg/kg) in rats induced a
significant elevation in the activities of urea and creatinine as compared to normal
control. This significant elevation was
attributed to the nephrotoxic effect of venom. This was in agreement with the
findings of Rahmy et al. (1992 & 1995b)
and Yaguchi et al. (1996). They mentioned that serious renal complications in case of
Naja haje and Cerastes cerastes
envenomation lead to impairment of the
excretory function of the kidney.
Significant elevation in serum urea
recorded in this work may also be attributed to an increase of nitrogen retention and / or
due to corrupted renal function (Abdel Nabi
1993). As for the effect on heart enzymes, the
present study indicated that the LD50 (27.69
µg/mouse) of native Echis pyramidum
venom caused a highly significant increase of Lactate Dehydrogenase (LDH), Creatine
Phosphokinase (CPK) and Creatine
Phosphokinase isoenzyme (CK-MB)compared to the normal control. The
obtained results are in agreement with those
previously reported by other investigators.
Aguiyi et al. (2001) reported an elevation in the levels of Lactate Dehydrogenase (LDH)
and Creatine Phosphokinase (CPK)
following administration of Echis
carinatus venom. Also, Fernando et al.
(1989) reported that B. asper venom caused
serum AST, LDH and CPK to increase significantly, the highest peak being
observed at 3hr in the cases of AST and
CPK, and at 6hr in the case of LDH. The
CPK isoenzymes, samples taken at 3hr showed a prominent increase in isoenzyme
CK-MM with a slight increase in CK-MB.
Moreover, Sofer et al., (1991) reported that the enzymatic activity of Creatine kinase -
MB isoenzyme and total Creatine
Phosphokinase (CPK) were elevated following envenomation by the scorpion
Leiurus quinquestriatus in children. They
speculated that the myocardial lesions are
too small to cause heart failure in most cases, but they may account for the
cardiovascular changes frequently seen in
scorpion envenomation. This assumption was also confirmed by the findings of
Hering et al., (1993) and Correa et al.,
(1997), who reported an increase in the
levels of CPK and LDH in patients stung by Tityus serrulatus scorpion and showed a
cardiomyopathy picture. Shaban and Hafez
(2003) reported that the intraperitoneal injection of a sublethal dose of Naja haje
venom (0.2mg/kg) in rats induced a
significant elevation in the activities of LDH and CK-MB as compared to normal
control.
Mohamed et al., (1981) reported the effect
of lethal doses of five venoms (Bitis
Influence of ….
- 327 -
arietans, Bitis gabonica, Dendroaspis,
Naja nigricollis and Naja haje) on the activities of transaminases, alkaline
phosphatase and lactate dehydrogenase in
albino rats. The activity of these enzymes were markedly increased in serum and
variably decreased in the liver, heart and
kidney after envenomation.
Measurement of enzyme activity in serum is of importance since it helps to assess the
state of the liver and other organs.
Normally serum transaminase levels are low, but after extensive tissue injury, these
enzymes are liberated into the serum and
the levels of serum transaminase, were
reported to be increased following damage to skeletal muscles, myocardial muscles
and liver (Harper et al. 1977). It was
suggested that, tissue destruction occurred in most of the organs secondary to venom
injections. The increase in enzymatic
activity of the serum might be due to release of the enzymes from liver, kidney
and heart (Metzler, 1977).
The rapid rise in ALT and AST activities
recorded in the present study 4 hr after venom envenomation may be attributed to
severe injuries and necrosis of hepatocytes
as well as to a nephrotoxic action of the venom as reported by Abdel Nabi (1993).
Also, Kadryov (1987) and Omran and
Abdel Rahman (1992), reported that lethal and sublethal doses of venom were capable
of stimulating stress reactions. Cortisol and
catecholamines are the main hormones
released in response to stress and increased levels of these hormones in victims
circulation may cause sever damage in
many vital organs proportional to the dose of the venom and the elapsid time. Organ
damage is followed by an increase in levels
of ALT, AST and ALP (Omran et al.,
1997). Rahmy and Hemmaid (2000 & 2001) reported that Cobra venom induced a
hepatotoxic action reflected by alteration in
the histological and histochemical patterns of the hepatic tissues. These alterations are
initiated at early stages of envenomation
and could indicate a disturbance in the functional activities of the liver during
envenomation.
Interestingly, the 1.5 kGy γ irradiated Echis
pyramidum venom caused a non significant
change (when used at a dose equal to that
used for the native venom; 27.69 µg/mouse) of ALT, AST, ALP, urea,
creatinine, LDH, CPK and CK-MB
compared to the normal control. This was in contrast to the native Echis pyramidum
venom (27.69 µg /mouse) that induced a
highly significant increase of ALT, AST,
ALP, urea, creatinine, LDH, CPK and CK-MB compared to the normal control or the
1.5 kGy γ irradiated Echis pyramidum
venom. After injection of muscle directly, we found
that ½ LD50 (14 µg / 50 µL) of native Echis
pyramidum venom induced a significant
increase of creatine kinase (CPK), released from the gastrocnemius muscles, compared
to normal control. In contrast, an equal dose
of 1.5 kGy γ irradiated Echis pyramidum venom showed a non significant increase of
creatine kinase when compared to normal
control. Moreover, the elevation in CPK levels observed with native Echis
pyramidum venom (14µg/50µl) was highly
significant compared to the 1.5 kGy γ
irradiated venom as well. These results indicate a decrease in the myotoxicity of the
γ irradiated Echis pyramidum venom.
Gutiérrez and Lomonte (1995) reported that myotoxic effect of venoms of the genus
Bothrops is particularly important, not only
because it may lead to permanent tissue loss, disabling the victim, but also because
it may induce severe cutaneous lesions on
the animals chronically exposed to the
venom during the immunization process. Souza et al. (2002) investigated the ability
of gamma radiation from 60
Co (2 kGy) to
attenuate the toxic effects of Bothrops
jararacussu venom on mouse
neuromuscular preparations in vitro. It was
concluded that 60
Co gamma radiation is
able to abolish both the paralyzing and the myotoxic effects of Bothrops jararacussu
venom on the mouse neuromuscular
junction. These findings support the hypothesis that gamma irradiation could be
an important tool to improve antisera
production by reducing toxicity while preserving immunogenicity.
Irradiation of crotoxin was shown to result
in its aggregation and generation of low
molecular weight breakdown products
Adel El-Missiry ….et al
- 328 -
(Nascimento and Rogero 1995). The
aggregates presented no toxicity, no phospholipase activity and no ability to
promote creatine kinase (CPK) release into
muscle tissue. Histological analysis of gastrocnemius
muscles subjected to native Echis
pyramidum venom revealed a large number
of muscle fibers drastically affected by the formation of dense clumps of
hypercontracted myofibrils alternating with
areas of cytoplasm apparently devoid of myofibrils. On the other hand, histological
analysis of gastrocnemius muscles
subjected to γ irradiated venom revealed a
large number of fibers with normal appearance and only few muscle cells
presenting edema in the connective tissue.
Injection of a sublethal venom dose in rats produced severe degeneration of muscle
fibers and loss of striations, also,
hemorrhage and extravasated red blood cells were seen in between the myocardial
bundles as recorded for several venoms by
Tu and Homma (1970), Rahmy et al.
(1995a & b) and Hanafy et al. (1999). In the present work the hemorrhage observed
in between the muscle receiving the native
venom may be due to increased intravascular tension or venous congestion
(Willoughly, 1960).
Collectively, the present data support the conclusion that gamma radiation is an
effective venom-detoxification method
which could help solve the chronic
problems of anti ophidic sera production. Results from the double immunodiffusion
test revealed a similar reactivity for native,
1 kGy, 1.5 kGy and 3 kGy γ irradiated Echis pyramidum venoms against a
commercial polyvalent Egyptian antivenin.
The visible lines obtained in the
immunodiffusion reactions were identical and join smoothly at the corners, indicating
that there was no change in their antigenic
reactivity. These results demonstrate that the ability of the venom antigens to react
with its corresponding antibodies was
maintained in spite of being exposed to radiation doses of 1 kGy, 1.5 kGy and 3
kGy.
The recorded results are in agreement with
those concluded by Shaban et al., (1996).
They indicated that there was no change in
antigenic reactivity with antibodies determinants. Also Shaban (2003) reported
that immunodiffusion studies revealed
identity between γ irradiated (15 kGy) and native Naja haje and Cerastes cerastes
venoms since the antigenic response was
not changed as judged by the capacity of
irradiated venoms to react with horse polyvalent antivenin. Moreover, Bennacef-
Heffar and Laraba-Djebari (2003) showed
that the effect of gamma irradiation on the venom of Vipera lebetina (one of the two
widespread snakes in Algeria). Vipera
lebetina venom was irradiated with two
doses of gamma rays (1 and 2 kGy) from a 60
Co source, and the venom's toxic,
enzymatic, and structural properties were
analyzed. Intraperitoneal injection of the irradiated venoms (100–500 µg/20 g mouse
body mass) revealed a significant decrease
in its toxicity. Irradiated venoms with 1 and 2 kGy doses were four and nine times less
toxic, respectively, than the native venom.
Moreover, the caseinolytic, amidolytic,
esterasic, and coagulative activities venom were reduced following irradiation only
phospholipase A2 activity was abolished in
the irradiated venom with a dose of 2 kGy. Both chromatographic and electrophoretic
profiles of the irradiated venom were
drastically changed as compared with that of the native venom. The gamma rays
detoxified venom was then used for active
immunization, and the presence of antibody
in the immune sera was detected by ELISA. The immunogenic properties of the
irradiated venom were preserved and the
antisera obtained were able to neutralize the toxic effect of Vipera lebetina native
venom. These results indicate that
irradiation of Vipera lebetina venom with a
dose of 2 kGy can promote a significant detoxification while keeping the
immunological properties intact).
Irradiation of Vipera lebetina venom with a dose of 2 kGy can promote a significant
detoxification while keeping the
immunological properties intact. Furthermore, Nascimento et al. (1998)
reported that ionizing radiation is able to
detoxify several venoms, including snake
venoms, without affecting their
Influence of ….
- 329 -
immunogenic properties significantly.
However, Kume and Matsuda (1995), studying the radiation induced changes in
the structural and antigenic properties of
egg albumin and bovine serum albumin, suggested that the main part of
conformation-dependent antigenic
structures (conformational epitopes) is
easily lost by radiation, but some antigenicity, which is mostly due to the
amino acid sequence-dependent antigenic
structures (sequential epitopes) remains, even at high doses.
Finally, both antivenins recognized Echis
pyramidum venom when submitted to
protein blotting, but the anti 1.5 kGy γ irradiated venom show a higher intensity
bands than the antivenin raised against
native Echis pyramidum venom. In spite of has less neutralizing activity (native
neutralize 50 LD 50, 1.5 kGy γ irradiated
neutralize 40 LD 50), this indicate that antibodies were formed not only for toxic
fraction but also for non toxic fraction.
These results agree with those of Clissa et
al. (1999) who reported that antivenin raised against the native venom and the 2
kGy γ irradiated venom recognized equally
all the bands present on the native Crotalus
durissus terrificus venom, but the ELISA
titre of recognition for each toxin was
higher for the serum anti-2 kGy γ irradiated venom than for the serum anti-native
venom. In order to ensure that the
improvement in immunogenicity of 2 kGy γ
irradiated venom was due to the presence of aggregates and not to minor amounts of
intact proteins left after irradiation, they
produced an antiserum against native Crotalus durissus terrificus venom
following an immunization protocol in
which the amount of intact protein injected
was similar the amount of intact protein present in the 2 kGy γ irradiated venom.
The antiserum obtained in this way had a
low ELISA titre and low neutralizing ability (Clissa et al. 1999).
In conclusion irradiation of the whole Echis
pyramidum venom with 1.5 kGy reduced its lethality 12.7 times while a higher dose
of radiation (3 kGy) destroyed practically
all the venom toxicity though keeping its
immunogenicity. However, the 1.5 kGy
dose was shown to be the best radiation
dose to promote venom detoxification without significantly affecting its
immunogenicity. Thus, our present results
confirm the conclusion that γ radiation is a suitable way to detoxify the Echis
pyramidum venom without affecting its
immunogenicity provided that a proper
dose is used. Moreover, these neutralization studies on
the antivenin raised against the 1.5 kGy γ
irradiated venom demonstrate the ability of the detoxified venom to be used as an
alternative to crude venom in immunizing
horses even in the initial doses of
immunization before immunity build up.
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Influence of ….
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دراسة التغيرات المناعية والكيميائية الحيوية نتيجة لتأثير االشعاع الجامى على
الحية الغريبة
**يحذ سعبء يحذ, *** عصذ عجذ انغالو شعجب , * عبدل عبل انغيش
*يبل اثشايى يصطف عجذ انحيذ , ** بديخ يحذ عجذهللا , * أحذ عجذ انجبعظ
كهيخ انطت عبيعخ عي شظ, يخ يشكض انجحس انطج*
كهيخ انعهو عبيعخ عي شظ**
يئخ انطبقخ انزسيخ –انشكض انقي نجحس ركنعيب االشعبع ***
يذف زا انجحش ان رحضيش عى يضعف أي ي انبحيخ انغيخ فعبل ي انبحيخ انبعيخ ثزعشيض عى انحيخ
.اي ف رحضيش االيصبل انضبدح انغشيجخ ان االشعبع انغبي العزخذ
قذ اشزم زا انجحش عه دساعخ انزغيشاد انكييبئيخ انحييخ انبرغخ ي اعزخذاو انغى انضعف يقبسخ يع
.انغى انخبو
كب رى اعزخذاو انغى انضعف انز يزى اخزيبس ف رحضيش انصم ف االسات
:يزى رهخيص انزبئظ كبالر
أ ز انغجخ قذ صادد يع صيبدح انزشعيع انغبي يع μg 27.79 عشعخ عيخ انحيخ انغشيجخ رضم عذ ا. 1
نكم فأس μg 353.23كبذ انغيخ kGy 1.5اعزخذاو عشعخ اشعبعيخ
فب انخطط. رغشثخ االزشبس انضدط أضحذ أ ال رغيش ف انصفبد انبعيخ ثبعزخذاو االشعبع انغبي . 2
.انبعيخ كبذ اضحخ ظبشح يع كم انغعبد
ثبنغجخ الخزجبس انطجع انبع ف قيبط يعبدنخ يكبد انغى أضحذ انزبئظ أ كم ي انصم انضبد نغى . 3
انحيخ انغشيجخ انصم انشعع نغى انحيخ انغشيجخ يحزيب عه أعغبو يضبدح نغيع عضيئبد انغى انخبو نك
.ق ف كيخ األعغبو انضبدح ف كهيب بك فش
(ALT &AST)ثبنغجخ نزأصيشاد انكيبئيخ انحييخ ي بحيخ ظبئف انكجذ عذ أ اضيبد انكجذ انبقهخ ناليي . 4
أيضب انفعفبريض انقه قذ صادد صيبدح راد دالنخ احصبئيخ ف انغعخ انعبيهخ ثبنغى األصه يقبسخ
. kGy 1.5ثبنغعخ انضبثطخ انغعخ انشععخ ثبالشعبع
. انضيبدح ف انغعخ انشععخ نيغذ راد دالنخ احصبئيخ يقبسخ ثبنغعخ انضبثطخ
شاد انكييبئيخ انحييخ ي بحيخ ظبئف انكهيخ فقذ أضحذ أ انضيبدح ف انجنيب أيضب ثبنغجخ نهزأصي. 5
انكشيبريي راد دالنخ احصبئيخ ف انغعخ انعبيهخ ثبنغى االصه يقبسخ ثبنغعخ انضبثطخ انغعخ
ثبنقبسخ ثبنغعخ صيبدح ف انغعخ انشععخ نيغذ راد دالنخ احصبئيخ kGy 1.5انشععخ ثبنشعبع
.ارا انزشعيع انغبي قهم ي انزأصيش انغ نغى انحيخ انغشيجخ عه انكهيخ. انضبثطخ
اضيى انهكزيذ دييذسعييض اضيى كشيبري كيبص )ثبنغجخ نهزأصيشاد انكييبئيخ انحييخ ي بحيخ ظبئف انقهت . 6
د دالنخ احصبئيخ ف انغعخ انعبيهخ ثبنغى االصه يقبسخ قذ صادد صيبدح را( انشبث االضي أو ث
ارا انزشعيع انغبي . نى رغغم ا رغييش احصبئ kGy 1.5ثبنغعخ انضبثطخ انغعخ انشععخ ثبنشعبع
.قهم ي انزأصيش انغ نغى انحيخ انغشيجخ عه انقهت
شكييظ نهفأس أضح أ اضيى كشيبري كيبص قذ صاد ف انزأصيش انغ نغى انحيخ انغشيجخ عه عضهخ انغبعز. 7
انجالصيب كبذ ز انضيبدح راد دالنخ احصبئيخ ف انغعخ انعبيهخ ثبنغى االصه يقبسخ ثبنغعخ انضبثطخ
ا انغعخ انشععخ نى رغغم رغيشاد احصبئيخ ثبنقبسخ ثبنغعخ kGy 1.5 انغعخ انشععخ ثبنشعبع
.ارا انزشعيع انغبي قهم ي انزأصيش انغ نغى انحيخ انغشيجخ عه عضالد . انضبثطخ
Adel El-Missiry ….et al
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أيضب ثزصيش عضهخ انغبعزشكييظ رحذ انيكشعكة أضحذ انصس أ رأصيش انغى انشعع ثبالشعبع
يضم انغعخ كب أفضم ثكضيش ي اعزخذاو انغى االصه نغى انحيخ انغشيجخ حز كبد أ يك kGy 1.5انغبي
.انضبثطخ
ثبنغجخ نزحضيش يصم يضبد نغى انحيخ انغشيجخ ف االسات أضحذ انزبئظ أ اخزجبس يعبدنخ انزأصيش انغ . 8
أ انصم انحضش ف حبنخ kGy 1.5نهغعزي انعبيهخ ثبنغى االصه انغعخ انعبيهخ ثبالشعبع انغبي
.ي بحيخ انغيخ kGy 1.5صم انحضش ف حبنخ انغى انشعع ثبنشعبع انغبي انغى االصه يغب رقشيجب ان
األفضم يع عى انحيخ انغشيجخ يك أ رضيم kGy 1.5رشيش زبئظ ز انذساعخ ان أ انغشعخ االشعبعيخ
انزبئظ ثيب كبذ نقذ أيك رحضيش يصم ي انغى األصه انغى انشعع . انغيخ رحزفع ثبنصفبد انبعيخ
ارا يك اعزخذاو انغى انضعف ف حق انخيل نزحضيش يصم فعبل أي نهقبيخ ي عى انحيخ . رقشيجب يزغبيخ
.انغشيجخ